Methods for nucleic acid amplification and sequence determination

Inactive Publication Date: 2006-02-02
FLUIDIGM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] Sequencing according to the invention comprises template-dependent nucleic acid synthesis. In a preferred embodiment, nucleic acid sequencing primers are exposed to amplicons having at least one primer binding site. A polymerase then directs the extension of the primer(s) in a template-dependent fashion in the presence of labeled nucleotides or nucleotide analogs. According to one aspect of the invention, amplicons are support-bound in a manner that allows unique optical identification of signaling events from the labeled nucleotide or nucleotide analogs as they are incorporated into the growing primer strand.

Problems solved by technology

While rolling circle amplification produces generally fewer amplicons than PCR, it still can result in the generation of many thousands of copies of the template.

Method used

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  • Methods for nucleic acid amplification and sequence determination
  • Methods for nucleic acid amplification and sequence determination
  • Methods for nucleic acid amplification and sequence determination

Examples

Experimental program
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Effect test

example 1

Creation of Anchored Amplicons Using Rolling Circle Amplification

[0070] The creation of an anchored amplicon from a linear nucleic acid template using rolling circle amplification involves (i) a circularization reaction in which the 5′ and 3′ ends of a linear nucleic acid template are ligated to form a circular nucleic acid template; (ii) a hybridization reaction in which a primer is hybridized to a single stranded circular template to create a circular template-primer hybrid; and (iii) an extension reaction in which the primer is extended by rolling circle amplification. The primer may contain one member of a binding pair that can bind to a binding partner that is attached to a solid support.

[0071] Briefly, a nucleic acid template is obtained from a cell or tissue, for example, using one of a variety of procedures for extracting nucleic acids, which are well known in the art. While the invention is exemplified below with synthetic oligonucleotides, the invention is not so limite...

example 2

Sequencing an Amplicon

[0086] This example demonstrates a method according to the invention in which a single nucleotide in a position in a nucleic acid molecule is identified. At least one sequencing primer is bound to an amplicon. The sequence of the primer in this example complementary to the 3′ linker binding site on the anchored primer, or, in effect, identical to at least a portion of the 3′ linker sequence. Alternatively, if linkers are not used, the primer may be complementary to any region of the circular template, preferably the 3′ end. The amplicon / primer complex is exposed first to a labeled nucleotide and then to an unlabeled nucleotide of the same type under conditions of, and in the presence of, reagents that allow template-dependent primer extension (FIG. 6). The signals of the labeled amplicons are then detected (FIG. 7).

Cycle Sequencing of Rolling Circle Products Bound to Streptavidin Tubes

[0087] After the primer bound rolling circle amplification described in ...

example 3

Analysis of Single Molecule Sequencing

[0090] Using a TIR Optical Setup such as that diagrammed in FIG. 9, images of a surface on which single molecule sequencing of an attached rolling circle amplified template has been performed are then analyzed for primer-incorporated U-Cy5. Typically, eight exposures of 0.5 seconds each are taken in each field of view in order to compensate for possible intermittency (e.g., blinking) in fluorophore emission. Software is employed to analyze the locations and intensities of fluorescence objects in the intensified charge-coupled device pictures. Fluorescent images acquired in the WinView32 interface (Roper Scientific, Princeton, N.J.) are analyzed using ImagePro Plus software (Media Cybernetics, Silver Springs, Md.). Essentially, the software is programmed to perform spot-finding in a predefined image field using user-defined size and intensity filters. The program then assigns grid coordinates to each identified spot, and normalizes the intensit...

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Abstract

The invention provides methods for sequencing a nucleic acid comprising conducting rolling circle amplification on a circular nucleic acid template, wherein the resulting amplicon is optionally anchored to a substrate in an individually optically resolvable manner, and performing a sequencing reaction.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 585,565, filed on Jul. 2, 2004, which is incorporated herein by reference.TECHNICAL FIELD OF THE INVENTION [0002] The invention relates to methods and devices for sequencing a nucleic acid, and more particularly, to methods and devices for preparing a nucleic acid template for high throughput single molecule sequencing. BACKGROUND OF THE INVENTION [0003] The completion of a consensus human genome sequence has given rise to inquiry into genetic differences within and between individuals as the basis for differences in biological function and dysfunction. For example, single nucleotide differences between individuals that give rise to single nucleotide polymorphisms (SNPs) can result in dramatic phenotypic differences. Those differences can be manifested in outward expressions of altered phenotype, can determine the likelihood that an individual will get a certain d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6806C12Q1/6869C12Q2565/501C12Q2531/125
Inventor LAPIDUS, STANLEYBUZBY, PHILIP
Owner FLUIDIGM CORP
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